HIV-1 protease inhibitors used in combination therapy are the most effective drugs in controlling HIV infection and propagation in vivo. There are currently four HIV-1 protease inhibitor drugs on the market and several in clinical trials. The high dosage, required in such treatment over a long time period, has heightened the problems of side effects and toxicity. Because HIV-1 protease belongs to the aspartic proteinase family, a probable source of the side effects is the cross inhibition of these drugs to human aspartic proteases. The inhibition of five known aspartic proteases by the HIV protease inhibitor drugs has been shown to be relatively low. However, the inhibition of the HIV protease inhibitors toward new human aspartic proteases, which were not discovered during the development of the current drugs, is not known. Lin and his colleagues have recently cloned the cDNA of four previously unknown aspartic proteases, napsin 1, napsin 2, aspin 1, and aspin 2. Knowledge of these new human aspartic proteases will be important for target specificity in the design of the new generation of HIV protease inhibitor drugs. These initial discoveries provide a unique opportunity for the understanding the inhibition of these new human enzymes by HIV protease inhibitor drugs and the physiological consequences of such inhibition. Furthermore, the active site specificities of these new enzymes may be understood in order to avoid cross reaction in the design of the new generation of HIV protease inhibitor drugs. The following specific aims are proposed to accomplish these goals. The genes for four new human proteases will be expressed and the proteins purified. The substrate specificity and subsite preferences of these human aspartic proteases will be studied. Finally, the inhibition of these four proteases by HIV-1 protease inhibitors will be tested and the relationships of inhibition to the toxicity of drug treatment will be assessed.